This grant proposal builds on our most important recent finding that the final cardiomyocyte population in mice is established during preadolescence, and not before birth, as previously believed. We showed that a precisely timed surge in circulating thyroid hormone (T3), occurring between postnatal day (P)10 and P12, precedes the P15 cardiomyocyte proliferation. In vivo inhibition of T3 biosynthesis in preadolescent mice prevents cardiomyocyte proliferation. Further, T3-treated cardiomyocytes show increases in DNA synthesis in vitro. An intriguing finding of these studies was that the potent cardiomyocyte mitogen IGF-1 and IGF-R/p-Akt signaling were increased at the time of the cardiomyocyte proliferation and, importantly, were suppressed by inhibition of T3 biosynthesis. Our new preliminary in vitro findings suggest that T3 increases the production of hydrogen peroxide (H2O2)-reactive oxygen species (ROS) and that very low concentrations of H2O2 transcriptionally activate IGF-1 gene expression and protein release from cardiomyocytes in culture. Further, low dose H2O2 activates mitogenic IGF-1/pAkt/pERK signaling in cardiomyocytes. Inhibiting H2O2 or IGF-1 generation prevented T3-mediated increase in the expression of genes critical for cell cycle reentry and mitosis. Thus our preliminary findings suggest that T3-dependent H2O2-ROS generation activates mitogenic IGF-1/pAkt/pERK signaling. To our knowledge, this mechanism has not been proposed before in cardiomyocytes, or indeed any other cell type. Therefore, the proposed studies are highly innovative. We hypothesize that T3 causes IGF-1 induction via the generation of H2O2 in vivo and this increase in IGF-1, a potent cardiomyocyte mitogen, plays an obligate role in inducing cardiomyocyte hyperplasia during preadolescence. We now offer 2 Specific Aims, with multiple proposed experiments in each Aim, to examine how T3 activates cardiomyocyte proliferation. Our proposed in vitro and in vivo studies using pharmacologic and genetic strategies in Specific Aim 1 examine the novel hypothesis that by increasing mitochondrial respiration/biogenesis, T3 increases H2O2-ROS generation, which then transcriptionally activates IGF-1 production. In Specific Aim 2, we examine whether IGF-1 is obligatory for cardiomyocyte proliferation in vivo in preadolescent hearts using multiple genetic mouse models and pharmacological approaches. Although IGF-1 is a known mitogen for cardiomyocytes during fetal heart development and during cardiac regeneration, its role in preadolescent heart growth, particularly as it relates to cardiomyocyte hyperplasia has not been explored. Understanding how cardiomyocyte replication is regulated in vivo could have important implications for cardiac regeneration in juvenile hearts.